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The Requirement of the Glutamic Acid Residue at the ThirdPosition from the Carboxyl Termini of the Laminin � Chainsin Integrin Binding by Laminins*

Received for publication, October 4, 2006, and in revised form, January 19, 2007 Published, JBC Papers in Press, February 15, 2007, DOI 10.1074/jbc.M609402200

Hiroyuki Ido1, Aya Nakamura1, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki,and Kiyotoshi Sekiguchi2

From the Laboratory of Extracellular Matrix Biochemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita,Osaka 565-0871, Japan

Laminins are the major cell-adhesive proteins in the base-ment membrane, consisting of three subunits termed �, �, and�. Theputative binding site for integrins has beenmapped to theG domain of the � chain, although trimerization with � and �chains is necessary for theGdomain to exert its integrin bindingactivity. Themechanism underlying the requirement of � and �chains in integrin binding by laminins remains poorly under-stood.Here, we show that theC-terminal region of the� chain isinvolved in modulation of the integrin binding activity of lami-nins. We found that deletion of the C-terminal three but nottwo amino acids within the �1 chain completely abrogatedthe integrin binding activity of laminin-511. Furthermore,substitution of Gln for Glu-1607, the amino acid residue atthe third position from the C terminus of the �1 chain, alsoabolished the integrin binding activity, underscoring the roleof Glu-1607 in integrin binding by the laminin.We also foundthat the conserved Glu residue of the �2 chain is necessary forintegrin binding by laminin-332, suggesting that the samemechanism operates in the modulation of the integrin bind-ing activity of laminins containing either �1 or �2 chains.However, the peptide segment modeled after the C-terminalregion of �1 chain was incapable of either binding to integrin orinhibiting integrin binding by laminin-511, making it unlikelythat the Glu residue is directly recognized by integrin. Theseresults, together, indicate a novel mechanism operating inligand recognition by laminin binding integrins.

Laminins are a family of glycoproteins present in the base-ment membrane (1–3). All laminins are large heterotrimericglycoproteins composed of�,�, and� chains that assemble intoa cross-shaped structure. To date, five � chains (�1–�5), three� chains (�1–�3), and three � chains (�1–�3) have been iden-tified, combinations of which yield at least 15 isoforms withdistinct subunit compositions (4). Laminins contribute to base-

ment membrane architecture and influence cell adhesion,spreading, and migration through binding to their cell surfacereceptors, particularly the integrin family of cell adhesionreceptors (5–9).Integrins play important roles in cell-matrix adhesion and

signaling events regulating proliferation and differentiation ofcells. Among the various integrin familymembers,�6�1,�6�4,�3�1, and �7�1 have been shown to be the major lamininreceptors expressed in many cell types (10). Binding sites forthese integrins have been mapped to the C-terminal globular(G)3 domain of the laminin � chains (6, 11–15). The G domainconsists of five tandemly repeated LG modules of �200 aminoacid residues, designated LG1 through LG5. By analogy withthe identification of the Arg-Gly-Asp (RGD) cell-adhesivemotif in fibronectin, many attempts have beenmade to identifyspecific sequences mimicking the integrin binding activity oflaminins. However, neither recombinant fragments of the Gdomain nor synthetic peptides modeled after the sequences inthe G domain have shown any significant cell-adhesive activitycomparable with that of intact laminins (15–18). Previously, wefound that deletion of the LG4-5 modules did not compromisethe ability of laminin-5114 (�5�1�1) to bind �3�1/�6�1 inte-grins, but further deletion up to the LG3 module resulted inthe loss of its integrin binding ability (19). These results indi-cate that the LG3 module is required for integrin binding bylaminin-511, although LG4-5 modules are dispensable. Never-theless, LG3 or other LG modules of the �5 chain did notexhibit any significant activity to bind the �6�1 integrin whenrecombinantly expressed alone or in tandem with adjacentmodules (20), suggesting that G domain per se is not sufficientto recapitulate the integrin binding activity of laminins.Despite the importance of the G domain in the � chain, sev-

eral lines of evidence indicate that heterotrimerization with �and � chains is required for laminins to exert their integrinbinding activities. Thus, the “E8 fragment” produced by briefelastase digestion ofmouse laminin-111 retained almost the fullactivity of the parentalmolecule tomediate integrin-dependentcell-substratum adhesion but lost its activity when its �1 chain* This study was partly supported by Grants-in-Aid for Scientific Research

15370055 (to K. S.) and the National Project on Protein Structural and Func-tional Analyses from the Ministry of Education, Science, Sports, and Cultureof Japan. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Tel.: 81-6-6879-8617; Fax:

81-6-6879-8619; E-mail: [email protected].

3 The abbreviations used are: G domain, globular domain; mAb, monoclonalantibody; BSA, bovine serum albumin; TBS, Tris-buffered saline; LG, lamininG-like; GST, glutathione S-transferase; LN, laminin.

4 A new nomenclature of laminin isoforms (51) has been used throughout thispaper: laminin-311, laminin-�3�1�1 (also designated laminin-6); laminin-332,laminin-�3�3�2 (laminin-5); laminin-511, laminin-�5�1�1 (laminin-10).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 15, pp. 11144 –11154, April 13, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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fragment containing LG1–3 modules was separated from thedisulfide-linked �1-�1 dimer fragment (21). Cell adhesiveactivity was restored to the �1 chain fragment upon in vitrorecombination and refolding with the �1-�1 dimer (21, 22).The importance of heterotrimerization with the � and � chainsin the integrin binding activity of laminins has also been dem-onstrated with the recombinant E8 fragment of laminin-332,which was reconstructed by combining the �3 chain fragmentcontaining LG1–3 modules with the disulfide-bonded �3-�2dimer segment (23). These results indicate that not only the Gdomain of the � chain but also heterotrimerization of the �chainwith the� and� chainswithin their coiled-coil domains isrequired for integrin binding by laminins, although the molec-ular basis of the requirement for the � and � chains remains tobe elucidated.To address the role of � and � chains in integrin binding by

laminins, we focused on the putative interface between the Gdomain of the � chain and the heterotrimerized coiled-coildomains. The coiled-coil domain of mouse laminin-111 hasbeen visualized by electron microscopy as cylinders (24–26);thus, it is conceivable that the C-terminal regions of the� and �chains are in close proximity to and possibly interact with theGdomain, thereby regulating the integrin binding activity of theG domain. Given the extension of nine amino acids in the �1chain against only one amino acid in the�1 chain, both ofwhichare C-terminal to the conserved disulfide bond between the �1and �1 chains (Fig. 1A), we focused our attention on the role ofthe C-terminal 9-amino acid extension of the �1 chain in inte-grin binding by laminin-511, a potent ligand for�6�1 and�3�1integrins. In the present study we examined the integrin bind-ing activities of a series of recombinant laminin-511 mutantswith deletions or substitutions within the C-terminal region ofthe �1 chain. Our results show that Glu-1607 at the third posi-tion from the C terminus is a prerequisite for laminin-511 rec-ognition by integrins.

EXPERIMENTAL PROCEDURES

Antibodies, Proteins, and Peptides—mAbs against the humanlaminin �5 chain (5D6; Ref. 27), the human laminin �1 chain(C12SW), and the human laminin �3 chain (2B10) were pro-duced in our laboratory. The function-blocking mAb againstintegrin �6 subunit (AMCI 7-4) was kindly provided by Dr.Masahiko Katayama (Eisai Co., Ltd, Tsukuba, Japan) (28).Hybridoma cells secreting the function-blocking mAb againstthe integrin �1 subunit (AIIB2), developed by Dr. CarolineDamsky (University of California, San Francisco, CA), wereobtained from the Developmental Studies Hybridoma Bank(University of Iowa, IA). The function-blocking mAb specificfor integrin �5 subunit (8F1) was produced in our laboratory(29). Horseradish peroxidase-conjugated anti-His6 and anti-FLAG mAbs were purchased from Qiagen and Sigma, respec-tively. A rabbit horseradish peroxidase-conjugated anti-hem-agglutinin tag polyclonal antibody was purchased from QEDBioscience (San Diego, CA). A polyclonal antibody against theACID/BASE coiled-coil peptides was generously provided byDr. Junichi Takagi (Institute for Protein Research, Osaka Uni-versity). Anti-GST mAb and horseradish peroxidase-conju-gated streptavidin were purchased from Zymed Laboratories

Inc. (San Francisco, CA). Human vitronectin was purified fromhuman serumaccording toYatohgo et al. (30).Human laminin-332 was purified from the conditioned medium of MKN45human gastric carcinoma cells. The synthetic peptidesmodeledafter the �1 C-terminal sequence (NTPSIEKP; designated as�1C peptide) and its mutant form (NTPSIQKP; designated as�1C(EQ) peptide) were purchased from Biologica (Nagoya,Japan).Construction of Expression Vectors—Soluble, clasped �6, �3,

and �1 integrin expression vectors were prepared as describedpreviously (10, 20). �V and �3 integrin expression vectors weregenerously provided by Dr. Junichi Takagi (31, 32). Expressionvectors for human laminin �5 chain lacking LG4-5 modules(pcDNA-�5�LG4-5), LG3–5 modules (pcDNA-�5�LG3–5),�1 chain (pCEP-�1), and �1 chain (pcDNA3.1-�1) were con-structed as described (19, 33). Expression vectors for the lami-nin �1 chain lacking the C-terminal 8 amino acids (�1�8AA), 3amino acids (�1�3AA), 2 amino acids (�1�2AA), or 1 aminoacid (�1�1AA) and that encoding the mutant �1 chain withsubstitution of Gln for Glu-1607 (�1EQ) were prepared as fol-lows. cDNAs encoding �1�8AA, �1�3AA, �1�2AA, �1�1AA,and �1EQ were amplified by PCR using pcDNA3.1-�1 as atemplate. PCR products were digested with BbvCI and XbaIand inserted into the corresponding restriction sites ofpcDNA3.1-�1. The PCR primers used were 5�-ACAGGCTGC-TCAAGAAGCCG-3� (forward primer of �1�8AA, �1�3AA,�1�2AA, �1�1AA, and �1EQ), 5�-AGCTTCTAGACTAGAA-GCAGCCAGATGG-3� (reverse primer of �1�8AA), 5�-AGC-TTCTAGACTAAATGGACGGGGTGTTG-3� (reverse pri-mer of �1�3AA), 5�-AGCTTCTAGACTATTCAATGGACGG-GGTG-3� (reverse primer of �1�2AA), 5�-AGCTTCTAGAC-TACTTTTCAATGGACGG-3� (reverse primer of �1�1AA),and 5�-AGCTTCTAGACTAGGGCTTCTGAATGGAC-3�(reverse primer of �1EQ).

Expression vectors for the E8 fragment of laminin-511, a het-erotrimer of the truncated �5, �1, and �1 chains (designated�5E8,�1E8, and �1E8, respectively), and itsmutant formswereprepared as follows. cDNAs encoding �5E8 (Ala2534–Ala3322),�1E8 (Leu1561–Leu1786), and �1E8 (Asn1364–Pro1609) wereamplified by PCR using pcDNA-�5, pCEP-�1, andpcDNA3.1-�1 as templates. His6 tag (for �5E8), hemagglutinintag (for �1E8), and FLAG tag (for �1E8) sequences were addedby extension PCRwith a HindIII site at the 5� end and an EcoRIsite at the 3� end. PCR products were digested with HindIII/EcoRI and inserted into the corresponding restriction sites ofthe expression vector pSecTag2B (Invitrogen). cDNAsencoding �1E8 mutants were amplified by PCR, and PCRproducts were inserted into the �1E8 expression vectorusing the following pairs of primers; 5�-AATGACATTCTC-AACAACCTGAAAG-3� (forward primer of E8-�1�3AAand E8-�1EQ), 5�-CTAAATGGACGGGGTGTTGAAG-3�(reverse primer of E8-�1�3AA), and 5�-CTAGGGCTTCTG-AATGGACGGGGTG-3� (reverse primer of E8-�1EQ).Expression vectors for human laminin �3 chain lacking

LG4-5 modules (nucleotides 1–4128), �3 chain, �2 chain, and�2 chainmutants were prepared as follows. A full-length cDNAencoding the �3 subunit lacking LG4-5 modules (GenBankTMaccession number NM_000227) was amplified by reverse tran-

Role of Laminin �1 Chain in Integrin Binding

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scription-PCR as a series of �1-kilobase fragments. Aftersequence verification, error-free cDNA fragments were ligatedin tandem, and the resulting cDNA of the �3 chain lackingLG4-5 modules was inserted into the expression vectorpcDNA3.1 (Invitrogen). A full-length cDNA encoding the �3subunit (GenBankTM accession number NM_001017402) waspurchased fromOpenBiosystems (Huntsville, AL) and insertedinto the expression vector pcDNA3.1. A partial cDNA encod-ing the �2 subunit (GenBankTM accession number NM_005562)was purchased from Open Biosystems, and the remaining por-tion of the cDNA was amplified by reverse transcription-PCR.After sequence verification, error-free cDNA fragments wereligated in tandem to construct a cDNA encompassing thewhole open reading frame, and the resulting cDNA wasinserted into the expression vector pcDNA3.1. cDNAs encod-ing �2mutants were amplified by PCR, and PCR products wereinserted into the �2 expression vector. The list of primersequences is available upon request.Expression vectors for GST fusion proteins containing

FNTPSIEKP (GST-�1(8AA)), QVTRGDVFT (sequence de-rived from vitronectin; GST-RGD), or QVTRGEVFT (GST-RGE) were constructed by inserting cDNA fragments encodingthe individual peptide sequences into the EcoRI/NotI restric-tion sites of pGEX4T-1 (Amersham Biosciences). The insertcDNAs were amplified by overlap extension PCR. The PCRprimers used were 5�-AATTGAATTCTTCAACACCCCGT-CCATTG-3� and 5�-ATATATATGCGGCCGCCTAGGGC-TTTTCAATGGACGG-3� (for GST-�1(8AA)), 5�-AATTG-AATTCCAAGTGACTCGCGGGGATG-3� and 5�-ATA-TATATGCGGCCGCCTAAGTGAACACATCCCCGCG-3�(for GST-RGD), and 5�-AATTGAATTCCAAGTGACTCG-CGGGGAAG-3� and 5�-ATATATATGCGGCCGCCTAA-GTGAACACTTCCCCGCG-3� (for GST-RGE).Expression and Purification of Recombinant Proteins—Re-

combinant laminin-511 mutants were produced using theFree-StyleTM 293 Expression system (Invitrogen) and purifiedfrom conditioned media as described previously (19). Recom-binant laminin-311 and laminin-332, both lacking LG4-5mod-ules, and their mutants were produced using the same expres-sion system. The resultant conditioned media were applied toimmunoaffinity columns conjugated with an anti-human lami-nin �3mAb 2B10. The columns were washed with TBS (50mMTris-HCl, pH 7.4, 150 mM NaCl), and bound laminins wereeluted with 0.1 M triethylamine, neutralized, and dialyzedagainst TBS. Recombinant �6�1, �3�1, and �V�3 integrinswere produced using the same expression system and purifiedfrom the conditionedmedia using anti-FLAG columns (Sigma)described previously (20).The recombinant E8 fragment of laminin-511 and its

mutants were produced using the Free-StyleTM 293 Expressionsystem. The conditioned media were applied to nickel nitrilo-triacetic acid affinity columns (Qiagen), and bound proteinswere eluted with 200 mM imidazole. The eluted proteins werefurther purified on anti-FLAG columns (Sigma). After dialysisagainst TBS, the purity of these recombinant proteins was ver-ified by SDS-PAGE followed by Coomassie Brilliant Blue stain-ing or immunoblotting with anti-His tag (for �5E8), anti-hem-agglutinin tag (for �1E8), or anti-FLAG tag (for �1E8) mAbs.

GST fusion proteins (GST-�1(8AA), GST-RGD, and GST-RGE) were induced in Escherichia coli with 0.1 mM isopropyl-�-D-thiogalactopyranoside and purified on glutathione-Sepha-rose 4B columns (AmershamBiosciences) after lysis of the cellsby sonication. Bound proteins were eluted with 50 mM Tris-HCl, pH 8.0, containing 10 mM glutathione. After dialysisagainst TBS, the purity of these recombinant proteins were ver-ified by SDS-PAGE followed by Coomassie Brilliant Blue stain-ing or immunoblotting.Binding Assays for �6�1, �3�1, and �V�3 Integrins—Solid-

phase integrin binding assays of recombinant laminins, theirmutants, orGST fusion proteins were performed using purifiedrecombinant �6�1, �3�1, and �V�3 integrins. 96-well micro-titer plateswere coatedwith recombinant proteins for testing atthe indicated concentrations. The amounts of the recombinantproteins adsorbed on the plates were quantified with mAb 5D6(for laminin-511, E8, and their mutants), mAb 2B10 (for lami-nin-311, laminin-332, and their mutants), or anti-GST anti-body (for GST fusion proteins) to confirm the equality of theamounts of adsorbed proteins. After blocking with 3% BSA,plates were incubated with the 20 nM recombinant�6�1,�3�1,or�V�3 integrin in the presence of 5mMMn2� at 37 °C for 1 h.�6�1, �3�1, and �V�3 integrins were used without proteolyticunclasping, since the binding ofMn2� to the integrin can over-come the structural constraint imposed by an artificially intro-duced interchain disulfide bond (32). After washing with TBScontaining 5 mM Mn2� and 0.05% Tween 20, bound proteinswere quantified after sequential incubations with the biotiny-lated anti-ACID/BASE antibody and horseradish peroxidase-conjugated streptavidin (20). For integrin binding inhibitionassays using function-blocking mAbs against integrin subunitsor synthetic peptides, adhesive proteins coated on the microti-ter plates were incubated with �6�1 integrin in the presence ofmAbs (100 �g/ml) or �1C or �1C(EQ) peptide at 37 °C for 1 hbefore incubationwith biotinylated anti-ACID/BASE antibody.Bound integrins were detected as described above. For deter-mination of the apparent dissociation constants by saturationbinding assays, plates were coated with laminins (5 nM). Afterblocking with BSA, serially diluted �6�1 integrin was added tothe plates, and bound integrin was quantified. The apparentdissociation constantswere determined as described previously(34).Cell Adhesion Assay—Cell adhesion assays were performed

as described previously (19) using K562 human leukemia cellstransfectedwith cDNAencoding an�6 integrin subunit (kindlyprovided by Dr. Arnoud Sonnenberg, The Netherland CancerInstitute, Amsterdam). The cells were maintained inRPMI1640 supplemented with 10% fetal calf serum and usedwithout pretreatment with Mn2� to fully activate integrins.After fixation with formaldehyde and subsequent staining withDiff-Quik (International Reagents Corp., Kobe, Japan),attached cells were counted in three independent wells usingScion Image software (Scion Corp., Frederick, MD).

RESULTS

Deletion of the C-terminal Region of the �1 Chain AbrogatesIntegrin Binding Activity—To address the potential involve-ment of the C-terminal region of the �1 chain in integrin bind-




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ing by laminin, we produced amutant laminin-511 inwhich theC-terminal eight amino acids were deleted from the �1 chain(designated LN511-�1�8AA; see Fig. 1A). Themutant laminin-511 also lacked the LG4-5 modules that are required for �-dys-troglycan binding but not for integrin binding (19). A recombi-nant laminin-511 containing an intact �1 chain and the �5chain deleted of LG4-5 (designated LN511-�1) was used as acontrol laminin-511 throughout this study. The authenticity ofthe recombinant proteins as well as other mutant laminin-511proteins with deletions or amino acid substitutions (see below)was verified by SDS-PAGE under reducing and non-reducingconditions (Fig. 1B). Under reducing conditions, the recombi-nant proteins gave three bands upon Coomassie Brilliant Bluestaining, one corresponding to the �5 chain lacking LG4-5modules and two lower bands corresponding to the �1 chainand intact or mutant �1 chains, the latter specifically detectedby immunoblotting with a mAb against the �1 chain (Fig. 1B).Under nonreducing conditions, LN511-�1 and its deletionmutants gave a slowly migrating band near the top of the gel,confirming that they were purified as trimers of the �5, �1, and�1 chains. Solid-phase binding assays with the �6�1 integrinshowed that the control laminin-511 (LN511-�1) was fullyactive in binding to the �6�1 integrin, whereas the mutantlaminin-511 lacking the C-terminal eight amino acids of the �1chain (LN511-�1�8AA) exhibited only marginal activity com-parable with that of another mutant laminin-511 lacking theLG3–5 modules of the �5 chain (designated �LG3–5; Fig. 2A),which have been shown to be devoid of integrin binding activity(19). Because LN511-�1 and LN511-�1�8AAhave common�5and �1 subunits, the significant reduction in integrin bindingactivity would be ascribable to the deletion of C-terminal eightamino acids in the �1 chain, indicating its involvement in inte-grin binding by laminin-511.To narrow down the amino acid residues involved in integrin

binding, we produced a series of mutant laminin-511 withshorter deletions within the C-terminal region of the �1 chainand examined their integrin binding activities (Fig. 1A).Mutantlaminin-511 lacking the C-terminal 1 or 2 amino acids (desig-nated LN511-�1�1AA and LN511-�1�2AA, respectively)retained the integrin binding activity but were less active thancontrol laminin-511 (Fig. 2A). Reduced integrin binding affinityof these deletion mutants was confirmed by determination ofapparent dissociation constants by saturation integrin bindingassays; the estimated dissociation constants of LN511-�1�1AAand LN511-�1�2AA for �6�1 integrin were 2.4 � 0.41 and2.2 � 1.4 nM, respectively, �5-fold lower than that of controllaminin-511 (Kd, 0.43 � 0.1 nM) (Fig. 3). Further deletion up tothe third amino acid residue, i.e. Glu-1607, resulted in a signif-icant abrogation of the integrin binding activity of laminin-511.Adramatic lossof the integrinbindingactivityupondeletionof theC-terminal three but not two amino acids from the �1 chain indi-cated that Glu-1607 at the third position from the C terminusplayed a critical role in thepotent integrinbindingby laminin-511.

FIGURE 1. Recombinant laminin-511 and its mutant proteins with dele-tions or amino acid substitution in the �1 chain. A, schematic representa-tion of recombinant laminin-511 and the C-terminal amino acid sequences ofits �1 chain with deletions or amino acid substitution. Cysteines are circled inblack, and the disulfide bond is depicted by a broken line in the upper scheme.The C-terminal eight amino acid residues of the �1 chain are shaded. C-termi-nal amino acid sequences of intact �1 chain and its mutants are shown in thebox. The cysteine residues are underlined. LN511-�1, recombinant laminin-511 lacking LG4-5 modules but containing intact �1 chain; LN511-�1�8AA,LN511-�1 lacking the �1 C-terminal 8 amino acids; LN511-�1�3AA, LN511-�1lacking the �1 C-terminal 3 amino acids; LN511-�1�2AA, LN511-�1 lacking the�1 C-terminal 2 amino acids; LN511-�1�1AA, LN511-�1 lacking only the mostC-terminal amino acid of the �1 chain; LN511-�1EQ, LN511-�1 in which theglutamic acid residue (E) was replaced by glutamine (Q, closed box). B, purifiedLN511-�1 and its mutants were analyzed by SDS-PAGE on 4% gels underreducing (left and center panels) and non-reducing conditions (right panel)followed by Coomassie Brilliant Blue (CBB) staining (left and right panels) orimmunoblotting (IB) with a mAb against the laminin �1 chain (center panel).Under reducing conditions, each recombinant protein gave three bands, onecorresponding to the �5 chain and two lower bands corresponding to the �1

and �1 chains; the latter was detected by an mAb against the �1 chain. Undernonreducing conditions, they gave a single band migrating at �800 kDa,confirming that they were purified as trimers of the �5, �1, and �1 chains. Thepositions of molecular size markers are shown in the left margin.




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To address the role of the Glu-1607 in integrin binding bylaminin-511, we produced amutant laminin-511 in which Glu-1607 of the �1 chain was replaced with glutamine (designated

LN511-�1EQ; see Fig. 1A). As expected, the laminin-511 withE1607Q substitution was almost devoid of integrin bindingactivity (Fig. 2B), confirming the importance of Glu-1607 inintegrin binding by laminin-511.Integrin Binding Activity of the E8 Fragment of Laminin-511

and Its Mutant Forms—The failure in the disulfide bond for-mation between the �1 and �1 chains near the C terminus hasbeen shown to destabilize the conformation of the coiled-coildomain of laminin (26). Because the deletions and amino acidsubstitution introduced to the �1 chain were very close to theconserved cysteine residue near the C terminus, these muta-tions could compromise the disulfide bond formation between�1 and �1 chains, resulting in the destabilization of the coiled-coil domain of the mutant proteins and, hence, the abrogationof their integrin binding activities.To examine this possibility, we produced E8 fragments of

control and mutant laminin-511 consisting of three truncatedsubunits modeled after the E8 fragment of laminin-111 (Fig. 4),since the truncated fragments of �1 and �1 chains contain onlyoneCys residue, allowing us to assess the effect of themutationsintroduced to the C-terminal region of the �1 chain on thedisulfide bond formation between �1 and �1 chains. Theauthenticity of the resulting recombinant E8 fragments of lami-nin-511 was verified by SDS-PAGE and immunoblotting with amAb against the FLAG tag, which was added to the N terminusof the truncated �1 chain. Under reducing conditions, eachrecombinant protein gave three bands upon Coomassie Bril-liant Blue staining, one corresponding to the truncated �5chain and two lower bands corresponding to the truncated �1and �1 chains (Fig. 4B). Control and mutant E8 fragments gavetwo bands under nonreducing conditions, one correspondingto the truncated �5 chain and another corresponding to theheterodimer of the truncated �1 and �1 chains. Because thetruncated �1 and �1 chains contain only one Cys residue nearthe C terminus, these results confirmed that the disulfide bondformation between these two chains was not compromised bythe mutations introduced near the C terminus of the �1 chain.The E8 fragment of control laminin-511, designated E8-�1,

exhibited potent integrin binding activity (Fig. 4C). Saturationintegrin binding assays demonstrated that the Kd of E8-�1 forthe �6�1 integrin was 1.5 � 0.02 nM, roughly comparable withthat of the parental protein (i.e. LN511-�1). However, two E8mutants with either deletion of the C-terminal three aminoacids (E8-�1�3AA) or substitution of Gln for Glu-1607 (E8-�1EQ) were almost devoid of integrin binding activity, furtherconfirming the importance of Glu-1607 in integrin binding bylaminin-511. These results also demonstrated that the loss ofintegrin binding activity by deletion of the C-terminal threeamino acids or E1607Q substitution was not due to a defect indisulfide bond formation between the �1 and �1 chains at theirC termini, but rather, it was due to the critical role of the regioncontaining Glu-1607 in sustaining the active conformation ofthe G domain or to its involvement in integrin binding by com-prising a part of the integrin recognition surface. The integrinbinding to E8-�1 was strongly inhibited by function-blockingmAbs against integrin �6 and �1 subunits. It was completelyblocked by a combination of anti-�6 and anti-�1 mAbs (Fig.4D), confirming the integrin binding specificity of E8-�1.

FIGURE 2. Binding of �6�1 integrin to laminin-511 mutants. 96-wellmicrotiter plates were coated with increasing concentrations of the followinglaminin-511 mutants, blocked with BSA, and then incubated with 20 nM inte-grin �6�1. A, LN511-�1�1AA, LN511-�1�2AA, LN511-�1�3AA, LN511-�1�8AA, and �LG3–5. B, LN511-�1EQ and �LG3–5. LN511-�1 was used as apositive control. The amounts of bound �6�1 integrin were quantified asdescribed under “Experimental Procedures.” Each point represents the meanof triplicate assays and the S.D., respectively.

FIGURE 3. Titration curves of recombinant �6�1 integrin bound to lami-nin-511 mutants. The amounts of the integrins bound in the presence of 5mM EDTA were taken as nonspecific binding and subtracted as background.The results are the means of duplicate determinations. Dissociation constantsof recombinant �6�1 integrin toward laminin-511 mutants are shown in thebox.




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Reduced Cell-adhesive Activity of Laminin-511 with E1607QSubstitution—The importance of Glu-1607 in integrin bindingwas further examined by cell adhesion assays using K562 cellsstably transfected with �6 integrin (35, 36). To avoid the auxil-iary interactions of laminin-511 with �V�3 and other integrinsrecognizing the Arg-Gly-Asp sequences within domain IVa(also designated domain L4b) of the�5 chain (37), we examinedthe cell-adhesive activities of the E8 fragments of control andmutant laminin-511 proteins.Mutant E8 fragments either lack-ing the C-terminal three amino acids or having E1607Q substi-tution were almost devoid of cell-adhesive activity, whereas thecontrol E8 fragment was fully active in mediating cell adhesionof K562 cells expressing the �6�1 integrin (Fig. 5). ControlK562 cells untransfected with the �6 integrin did not adhere toany of the recombinant E8 fragments, confirming the specificityof the �6�1 integrin-dependent cell adhesion. These resultsfurther support our conclusion that the C-terminal region ofthe �1 chain, in particular Glu-1607 at the third position fromthe C terminus, played an important role in integrin binding bylaminin-511.

FIGURE 4. Production of recombinant E8 fragments of control andmutant laminin-511. A, schematic representations of recombinant laminin-511 and its E8 fragment. B, control E8 fragment (E8-�1) and its mutant pro-teins (E8-�1�3AA and E8-�1EQ) were analyzed by SDS-PAGE on 12% gelsunder reducing (left panels) and non-reducing (right panels) conditions fol-lowed by Coomassie Brilliant Blue (CBB) staining or immunoblotting (IB) witha mAb against the FLAG tag. The positions of molecular size markers areshown in the left margin. C, binding of �6�1 integrin to E8 fragments of lami-nin-511 and its mutant proteins. 96-well microtiter plates were coated with

increasing concentrations of LN511-�1 and E8 fragments of laminin-511 (E8-�1) and its mutant proteins (E8-�1�3AA and E8-�1EQ), blocked with BSA, andthen incubated with 20 nM �6�1 integrin. D, inhibition of integrin binding toE8-�1 using anti-integrin mAbs. 96-well microtiter plates were coated withE8-�1 (5 nM). �6�1 integrin (20 nM) was preincubated with function-blockingmAbs against integrin subunits and then added to the precoated wells. Theamounts of bound �6�1 integrin were quantified as described under “Exper-imental Procedures.” Each column and bar represents the mean of triplicateassays and the S.D., respectively.

FIGURE 5. Cell-adhesive activity of E8 fragments of laminin-511 and itsmutant proteins. K562 cells were incubated at 37 °C for 30 min on 96-wellmicrotiter plates coated with LN511-�1 and E8 fragments of laminin-511 (E8-�1) and its mutant proteins (E8-�1�3AA and E8-�1EQ). Adherent cells werefixed and stained as described under “Experimental Procedures.” A, repre-sentative images of wild type K562 and �562 cells expressing �6�1 integrinadhering to the substrates. Bar � 60 �m. B, cells adhering to the substrateswere counted as described under “Experimental Procedures.” Each point rep-resents the mean of triplicate assays and the S.D., respectively.




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The C-terminal Region of the �1 Chain Does Not Function asan Integrin-binding Site—The binding profiles of the �1�3AAand �1EQmutants of laminin-511 toward �6�1 integrin raisedthe possibility that the C-terminal region of the �1 chain couldcomprise part of the binding site for �6�1 integrin. To explorethis possibility, we expressed the C-terminal eight amino acidresidues of the �1 chain in bacteria as a GST fusion protein(GST-�1(8AA): Fig. 6A) and examined whether it could bind tothe �6�1 integrin. Neither GST alone nor GST-�1(8AA)showed any significant integrin binding activity, even at thecoating concentrations as high as 200 nM, although controlLN511-�1 bound a significant amount of the integrin even at 5nM (Fig. 6B), making it unlikely that the C-terminal peptidesegment of the �1 chain is directly recognized by the �6�1integrin. The failure of GST-�1(8AA) to bind to the �6�1 inte-grin could arise from the bulky GST domain, blockading directinteraction of the �1 peptide with the integrin, although theRGD- but not RGE-containing peptide segment fused to GSTexhibited significant binding activity toward the �V�3 integrin(Fig. 6C), making this possibility unlikely.

To further explore the possibility that the�1C-terminal pep-tide directly binds to the integrin, we examined whether a syn-thetic peptide modeled after the �1 C-terminal sequence wascapable of inhibiting integrin binding. Thus, binding of the sol-uble �6�1 integrin to immobilized LN511-�1 was determinedin the presence of increasing concentrations of the synthetic 8amino acid peptide derived from the C-terminal region of the�1 chain (designated �1C peptide) or its mutant form, in whichGlu-1607 was replaced by glutamine (�1C(EQ) peptide; Fig. 7)The �1C peptide did not exert any significant inhibitory effecton integrin binding to LN511-�1 even at 2.5 mM (125,000-foldmolar excess to the integrin added) as compared with the con-trol peptide with the E1607Q substitution. These results argueagainst the direct recognition of theC-terminal region of the �1chain by the integrin and support the possibility that the �1chain modulates the integrin binding activity of laminin-511through interactionwith theGdomain of the�5 chain, possiblystabilizing the conformation of the G domain active in integrinbinding.Glu-1607 Is Required for Laminin Binding by �3�1 Integrin—

Laminin-511 has been shown to serve as a potent ligand forboth �3�1 and �6�1 integrins (10, 34). Therefore, we nextexamined whether Glu-1607 was required for laminin-511binding to the �3�1 and �6�1 integrins. Solid-phase bindingassays using the �3�1 integrin demonstrated that �1�3AA and�1EQ mutant proteins were barely active in binding to the�3�1 integrin, exhibiting a �80% decrease when comparedwith control laminin-511 (Fig. 8A). Thus, Glu-1607 is requiredfor high affinity binding of laminin-511 to both �3�1 and �6�1integrins, underscoring its importance in laminin-511 recogni-tion by cognitive integrins.To extend the role of Glu-1607 in integrin binding to other

laminin isoforms containing the �1 chain, we examined inte-grin binding to laminin-311 (�3�1�1), which is also a ligand forthe �3�1 integrin (38). We expressed and purified laminin-311lacking LG4-5 modules of the �3 chain (designated LN311-�1)and its �1 mutants lacking the C-terminal three amino acids(LN311-�1�3AA) or having an E1607Q substitution (LN311-

FIGURE 6. Integrin binding activities of the C-terminal peptide segmentof the �1 chain. A, SDS-PAGE profiles of GST fusion proteins containing the�1 C-terminal 8 amino acids (GST-�1(8AA)), QVTRGDVFT (GST-RGD), or QVT-RGEVFT (GST-RGE) peptides. The RGD-containing peptide was modeled aftervitronectin. Proteins were stained with Coomassie Brilliant Blue. The positionof the 29-kDa molecular size marker is shown in the left margin. B, �6�1integrin binding activities of GST, GST-�1(8AA), and LN511-�1. 96-well micro-titer plates were coated with recombinant proteins at the indicated concen-trations and then incubated with 20 nM �6�1 integrin. The amounts of boundintegrin were quantified as described in under “Experimental Procedures.”C, �V�3 integrin binding activities of GST, GST-RGD, GST-RGE, and purifiedvitronectin. Each column and bar represents the mean of triplicate assays andthe S.D., respectively. The RGD-containing peptide fused to GST was equallyactive in binding to �6�1 integrin as vitronectin.

FIGURE 7. Inhibition of �6�1 integrin binding to laminin-511 by synthetic�1 peptides. �6�1 integrin (20 nM) was incubated on microtiter platescoated with 10 nM LN511-�1 in the presence of increasing concentrations of�1C (NTPSIEKP) and �1C(EQ) (NTPSIQKP) peptides for 1 h. Bound integrinswere quantified as described under “Experimental Procedures.” Each columnand bar represents the mean of triplicate assays and S.D., respectively.




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�1EQ) and examined their �3�1 integrin binding activities. Asexpected, LN311-�1 exhibited potent �3�1 integrin bindingactivity, whereas LN311-�1�3AA and LN311-�1EQ werealmost devoid it (Fig. 8B). These results are consistent with theconclusion that Glu-1607 is indispensable for �1-containinglaminins to exert their integrin binding activities.Glutamic Acid Residue at the Third Position from the C Ter-

minus of the �2 Chain Is Required for Integrin Binding byLaminin-332—The glutamic acid residue at the third positionfrom the C terminus is conserved between laminin �1 and �2chains (Fig. 9). The presence of the conserved glutamic acidresidue (Glu-1191) in the �2 chain raised the possibility thatthis Glu residue is also required for integrin binding of the �2chain-containing laminin, i.e. laminin-332 (�3�3�2). Toaddress this possibility, we expressed and purified laminin-332lacking LG4-5 modules (designated LN332-�2) and its mutantproteins either lacking the C-terminal three amino acids of the�2 chain (LN332-�2�3AA) or having E1191Q substitutionwithin the �2 chain (LN332-�2EQ) and examined their �3�1integrin binding activities. Recombinant LN332-�2 was foundto possess potent �3�1 integrin binding activity, comparable

with that of laminin-332 purified from a conditioned mediumofMKN45 human carcinoma cells (Fig. 9). However, neither �2chainmutants, LN332-�2�3AA and LN332-�2EQ, showed anysignificant binding to the �3�1 integrin. These results indicatethat Glu-1191 within the �2 chain is involved in integrin bind-ing by laminin-332 and provide evidence that the C-terminalregion of the � chains, particularly the Glu residue at the thirdposition from the C terminus, has a critical role in integrinbinding by laminins containing either �1 or �2 chains.

DISCUSSION

Despite accumulating evidence of the requirement of hetero-trimerization of the � chain with a disulfide-bonded dimer ofthe � and � chains in integrin binding by laminins (21–23), nostudies have before directly addressed the roles of � and �chains in integrin binding through production of mutant lami-nin isoforms with deletions and/or amino acid substitutions inthe � and/or � chains. In the present study we provide evidencethat three amino acids within the �1 C-terminal region, partic-ularly Glu-1607 at the third position from the C terminus, arerequired for the integrin binding activity of laminin-511. Thisconclusion is based on the following observations. First, dele-tion of the C-terminal three but not two amino acids within the�1 chain completely abrogated the integrin binding activity oflaminin-511, as was the case with substitution of Gln for Glu-1607 at the third position from the �1 C terminus. Second, the

FIGURE 8. Binding of the �3�1 integrin to laminin-511, laminin-311, andtheir mutant proteins. A, 96-well microtiter plates were coated with 10 nM

LN511-�1 and its mutant proteins, blocked with BSA, and then incubatedwith 20 nM �3�1 integrin. The amounts of bound �3�1 integrin were quan-tified as described under “Experimental Procedures.” B, binding of the �3�1integrin to laminin-311 mutants. LN311-�1, recombinant laminin-311 lackingthe LG4-5 modules but containing intact �1 chain; LN311-�1�3AA, LN311-�1lacking the �1 C-terminal 3 amino acids; LN311-�1EQ, LN311-�1 in which glu-tamic acid residue was replaced by glutamine.

FIGURE 9. Binding of the �3�1 integrin to laminin-332 mutant proteins.C-terminal amino acid sequences of intact �1 and �2 chains and the mutantforms of �2 chain are shown in the box. Cysteine and glutamic acid residuesconserved between �1 and �2 chains are underlined. The closed box repre-sents the substituted glutamine residue. LN332, human laminin-332 purifiedfrom the conditioned medium of MKN45 human carcinoma cells; LN332-�2, recombinant laminin-332 lacking the LG4-5 modules but containingintact �2 chain; LN332-�2�3AA, LN332-�2 lacking the C-terminal 3 aminoacids of �2 chain; LN332-�2EQ, LN332-�2 in which glutamic acid residue(E) was replaced by glutamine (Q). Each column and bar represents themean of triplicate assays and the S.D., respectively.




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C-terminal region of the �1 chain is required for not onlyLN-511 but also LN-311 to bind to integrins. Third, the Gluresidue at the third position from the C terminus is conservedfor both �1 and �2 chains, and deletion of the C-terminal threeamino acids within the �2 chain or substitution of Gln for Glu-1191 in the �2 chain completely abrogated the integrin bindingactivity of laminin-332.One of the likely explanations for the requirement of the �1

C-terminal region, particularly the Glu residue at the thirdposition from the C terminus, in integrin binding by lamininscould be that Glu-1607 comprises part of the integrin recogni-tion site of�1 chain-containing laminins (Fig. 10A). Because themetal ion bound to theMIDAS (metal ion-dependent adhesionsite) motif within integrins directly coordinates the side chainof acidic residues in ligands (39), acidic residues such as Asp orGlu are considered to act as key residues in ligands to interactwith integrins (40, 41). However, despite the importance of theGlu-1607 in integrin binding by �1 chain-containing laminins,the C-terminal peptide segment of the �1 chain fused to GSTand did not show any significant activity to bind the �6�1 inte-grin, and the synthetic peptide modeled after the C-terminalpeptide sequence of the �1 chain did not exert any significantinhibitory effect on integrin binding by laminin-511 even atconcentrations as high as 2.5 mM. These results argue stronglyagainst the possibility that the C-terminal segment of the �1chain including Glu-1607 functions as an integrin-binding site,althoughwe cannot exclude the possibility that the �1C-termi-nal segment comprises part of the integrin recognition site,

whose contribution to integrin binding by laminins is ratherauxiliary and not sufficient to be detected in direct integrinbinding or integrin binding competition assays. Another poten-tial explanation for the requirement of the C-terminal region ofthe �1 chain in integrin binding by laminins could be that dele-tion or replacement of Glu-1607 may result in instability inlaminin chain assembly and interferes with disulfide bond for-mation between � and � chains near their C termini. Becausethe conformation of the coiled-coil domain of laminin has beenshown to be destabilized by disruption of the C-terminal disul-fide bonds between � and � chains (26), deletion of the C-ter-minal three amino acids from the �1 chain or E1607Q substi-tution could impair �-� dimer formation through disulfidebonding, thereby leading to destabilization of the conformationof laminin-511 and, hence, inactivation of its integrin bindingactivity. However, SDS-PAGE analysis of purified E8-�1�3AAand E8-�1EQ under non-reducing conditions showed that adisulfide bond was spontaneously formed between truncated�1 and �1 chains (Fig. 4B), making this possibility unlikely.

The third explanation for the requirement of the C-terminalregion of the �1 chain in integrin binding is that theGlu residueat the third position from the C terminus of �1 chain directlyinteracts with the G domain and alters its local or global con-formation, thereby exposing the putative integrin-binding sitewithin the G domain that otherwise remains cryptic. A hypo-theticalmodel for the structure of the entireG domain has beenproposed (14) based on the three-dimensional structure ofLG4-5 modules of the �2 chain and the known length of inter-domain linkers (42). The model predicts that LG1–3 moduleshave the shape of a cloverleaf and that the coiled-coil domainadjacent to the LG1–3 modules seems to make direct contactwith the cloverleaf. This model together with the resultsobtained in this study raises the possibility that the Glu residuein theC-terminal region of� chains is in direct contactwith oneof the LG modules and alters either the local conformation ofindividual LG1–3 modules or the global configuration of thetandem array of LG1–3modules, thereby exposing the putativeintegrin-binding site within LG1–3 modules (Fig. 10B). Previ-ously, we demonstrated that the LG3 module is indispensablefor binding to the �6�1 integrin (19), although neither the LG3module alone nor a tandem array of LG1–3 modules was capa-ble of binding to the �6�1 integrin (20), suggesting that a tan-dem array of the LG1–3 modules per se is not sufficient torecapitulate the integrin binding activity of laminins. It istempting, therefore, to speculate that the integrin bindingactivity depends on both strictly regulated conformation ofLG1–3 modules and Glu-1607 of the �1 chain, the latter inter-acting with LG1–3modules and thereby stabilizing their activeconformation, so that deletion or replacement of the Glu-1607results in abrogation of the integrin binding activity of LG1–3modules through destabilization of their active conformation.Further investigation is needed to confirm this possibilitythrough defining the putative site within the G domain thatinteracts with Glu-1607.We also showed that Glu-1191 within the �2 chain, which is

located at the third position from the C terminus, is alsorequired for the integrin binding activity of laminin-332,extending the importance of the Glu residue at the third posi-

FIGURE 10. Schematic models for � chain-dependent recognition of lami-nin by integrin. In model A, the Glu residue within the C-terminal region ofthe � chain is directly involved in integrin binding by laminin as a criticalamino acid residue recognized by integrin. In model B, the C-terminal Gluresidue of the � chain interacts with the G domain and induces (or exposes)the putative integrin-binding site within the G domain through conforma-tional modulation. Alternatively, the C-terminal Glu residue may also com-prise part of the integrin recognition site, whose contribution to integrinbinding by laminins is rather auxiliary and not sufficient to be detected indirect integrin binding assays or integrin binding inhibition assays.




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tion from the C terminus to the �2 chain. Previously, a novelvariant �2 chain arising from alternative splicing of the 3� mostexon was identified and designated �2 transcript variant 2(GenBankTM accession number NM_018891; Refs. 43 and 44).This variant �2 chain is shorter than the normal �2 chain andlacks Glu-1191. It seems likely, therefore, that the laminin iso-form containing the variant �2 chain is defective in binding tothe �3�1 integrin due to the absence of Glu-1191 that isrequired for the integrin binding by laminin-332. The tran-script for the variant �2 chain was detected in cerebral cortex,lung, and distal tubules of the kidney (43). Generation of twosplice variants of laminin �2 chains with and without integrinbinding activity may be a novel mechanism modulating theinteraction of cells with the basement membrane containinglaminin-332 in these tissues.It is interesting to note that a novel � chain isoform, �3

(GenBankTM accession number NM_006059), also lacks theGlu residue that is conserved between the �1 and �2 chains(45). Furthermore, the C-terminal region of the �3 chain con-sists of only four amino acid residues after the conserved Cysresidue, significantly shorter than the corresponding regions ofother � chains. These differences between �3 and other �chains raise the possibility that the laminin isoform containingthe �3 chain is defective in binding to integrins. Recently, Yanand Cheng (46) reported, however, that the �3 chain forms aheterotrimer with �3 and �3 chains and serves as a ligand forthe �6�1 integrin at the apical ectoplasmic specialization in rattestes. Thus, the �3 chain-containing laminin may well retainintegrin binding activity despite the absence of the conservedC-terminal Glu residue in the �3 chain. Heterotrimerization ofthe �3 chain with �3 and �3 chains suggests that the laminincontaining the �3 chain may circumvent the loss of the Gluresidue, so that the G domain of laminin-333 can maintainactive conformation in the absence of the conserved Glu resi-due in the �3 chain. However, laminin-333 has been neitherpurified nor examined directly for its cell-adhesive and integrinbinding activities. It remains to be determined whether lami-nin-333 is capable of binding to �6�1 and other laminin-bind-ing integrins and, if so, how potent the binding is as comparedwith the binding of laminin-332 to �6�1 and other lamininbinding integrins.Little is known about the role of the laminin � chain in inte-

grin binding by laminins. Although the YIGSR sequence withinthe short armof the�1 chain has been proposed as an active sitethat mediates cell attachment, migration, and receptor binding(47–49), the biological significance of this sequence remains tobe confirmed. In contrast to the nine amino acid extensionC-terminal to the conserved Cys residue in �1 and �2 chains,theC-terminal region of the�1,�2, and�3 chains contains onlyone amino acid extension C-terminal to the conserved Cys res-idue, making it less likely that the C-terminal region of � chainsis directly involved in modulation of the integrin binding activ-ity of laminins, although we cannot yet exclude the possibility.Recently, Nishimune et al. (50) demonstrated that the laminin�2 chain, a component of the synaptic cleft at the neuromus-cular junction, binds directly to calcium channels that arerequired for neurotransmitter release frommotor nerve termi-nals. Furthermore, they showed that a C-terminal 20-kDa frag-

ment of the �2 chain, in particular leucine-arginine-glutamatetripeptide within this fragment, is necessary and sufficient forlaminin interactions with voltage-gated calcium channels, sug-gesting that the C-terminal region of the � chain functions asthe binding site for protein(s) other than integrins.In conclusion, our data showed that the Glu residue within

theC terminus of the �1 chain is necessary for binding to�6�1/�3�1 integrins and is potentially involved in maintenance ofthe active conformation of the G domain. It is interesting tonote that this Glu residue is highly conserved from nematodesto mammals, suggesting that molecular mechanisms underly-ing integrin binding to laminins are evolutionarily conservedthroughout metazoans. Our results provide new insights intothe modulatory role of the � chain in integrin binding bylaminins.

Acknowledgments—We thank Noriko Sanzen for establishing hybri-doma clones for laminins and purifying the mAbs, Dr. MasahikoKatayama for the mAb against integrin �6 subunit (AMCI 7-4), andDr. Caroline Damsky for the mAb against integrin �1 subunit(AIIB2). We also thank Dr. Arnoud Sonnenberg for providing theK562 cells expressing �6�1 integrin and Dr. Junichi Takagi for pro-viding the recombinant �1, �V, and �3 integrin expression vectorsand anti-ACID/BASE polyclonal antibody.

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Futaki and Kiyotoshi SekiguchiHiroyuki Ido, Aya Nakamura, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko

Chains in Integrin Binding by LamininsγCarboxyl Termini of the Laminin The Requirement of the Glutamic Acid Residue at the Third Position from the

doi: 10.1074/jbc.M609402200 originally published online February 15, 20072007, 282:11144-11154.J. Biol. Chem.

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